Project Summary/Abstract Breast cancer accounts for the second most cancer-related deaths in U.S. women despite the availability of improved treatment options and increased screening. A particularly aggressive subtype that represents a disproportionate number of these mortality cases is basal-like breast cancer (BLBC). This subtype frequently relapses and has a propensity to metastasize. Moreover, no directed therapies exist for BLBC. Hence, to improve outcomes for patients with this subtype, the identification of novel vulnerabilities in BLBC that would allow for the design and development of directed treatments is required. Strikingly, we find that BLBC cell lines, but not luminal cell lines, exhibit a severe sensitivity to suppression of iron-sulfur cluster (ISC) biosynthesis, a pathway that supports the function of at least 48 proteins involved in cellular processes such as energy metabolism, iron homeostasis, and DNA replication and repair. Moreover, suppression of NFS1, a key enzyme in the ISC biosynthetic pathway, prevents basal-like breast cancer metastasis to the lung. Thus, the identification of which downstream ISC containing proteins drive this sensitivity promises to elucidate pathway vulnerabilities in BLBC that could potentially lead to a targeted therapy for this subtype with a poor prognosis and limited treatment options. The proposed work in this fellowship will identify differentially required ISC containing proteins in BLBC and then will validate them using in vitro and xenograft models to investigate the effects of suppressing the proteins on tumor formation, growth, and metastasis. Validated targets will then be tested in the same models for synergy with current BLBC treatments. Preliminary data suggests that induction of genomic instability upon ISC biosynthesis suppression contributes to the proliferation defects observed in BLBC cell lines. Further experiments demonstrate that DNA Polymerase ? (POLE), the leading strand replicative polymerase, is one such differentially required protein. We hypothesize that BLBC is highly sensitive to POLE suppression due to a DNA repair defect in this tumor type that renders POLE activity critical for genomic integrity. To evaluate this hypothesis, complementary and independent approaches that analyze DNA damage signaling pathways, examine replication propagation and origin firing, and genetically dissect the role of POLE subunits will be employed. These approaches will provide a stringent characterization of the role of POLE in DNA damage repair pathways in BLBC. Collectively, our proposed work will identify and characterize ISC pathway vulnerabilities in BLBC from which novel targeted therapies could be developed.